Project Summary Acute myeloid leukemia (AML) is highly aggressive, and majority of AML patients eventually become refractory to chemotherapy and succumb to the malignancy. As etiology for AML is very heterogeneous, it is challenging to develop an effective approach to treat majority of AML patients. Adoptive cell therapy using chimeric antigen receptor (CAR)-expressing T cells is very successful for treating lymphocytic leukemia and lymphoma by targeting CD19, and has recently been approved by FDA for clinical use. However, the similar approach has not yet been extensively explored to achieve success for AML. A sorely unmet need is to develop new approaches to target AML to improve therapy. We have recently developed an innovative system to isolate antibodies that bind AML cells and enable the cognate CAR T cells to kill the cancer cells. Using this system, we successfully isolated nanobodies, which can bind epitopes with a single domain, from immunized llama. Two of the nanobodies specifically bind to a cell surface protease, which is expressed in AML cells from > 80% of AML patients. Notably, the CAR T cells targeting the cell surface protease potently and specifically eradicated AML cells in vitro and in AML cell line-derived xenograft. To control the potential on-target, off- tissue toxicity of the CAR T cells, we further developed a conditionally inducible CAR as well as bispecific and split CAR T cells to kill AML cells in vitro and in vivo. We hypothesize that the nanobody-directed CAR T system can eradicate AML patient derived xenografts (PDX), and this approach can be further developed to treat AML with tolerable toxicity. Two specific aims are proposed to test this hypothesis: Aim 1 will investigate the impact of the nanobody 157 (Nb157)-directed CAR T cells on eradicating primary human AML cells in preclinical models. Aim 2 will improve and evaluate the Nb157-directed CAR, in combination with other AML- associating antigen-specific split CAR T to increase AML-specific killing of the AML patient-derived leukemia cells, with reduced toxicity to normal cells. These studies will likely establish a novel and safe CAR system to controllably eradicate AML, paving the way to significantly improve AML therapy.